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Background Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. Methods Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. Results Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. Conclusions We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

Supplementary Data from Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas

Purpose: Hypothalamic or locally produced growth factors and cytokines control pituitary development, functioning, and cell division. We evaluated the expression of the chemokine stromal cell–derived factor 1 (SDF1) and its receptor CXCR4 in human pituitary adenomas and normal pituitary tissues and their role in cell proliferation.Experimental Design: The expression of SDF1 and CXCR4 in 65 human pituitary adenomas and 4 human normal pituitaries was determined by reverse transcription-PCR, immunohistochemistry, and confocal immunofluorescence. The proliferative effect of SDF1 was evaluated in eight fibroblast-free human pituitary adenoma cell cultures.Results: CXCR4 mRNA was expressed in 92% of growth hormone (GH)-secreting pituitary adenomas (GHoma) and 81% of nonfunctioning pituitary adenomas (NFPA), whereas SDF1 was identified in 63% and 78% of GHomas and NFPAs, respectively. Immunostaining for CXCR4 and SDF1 showed a strong homogenous labeling in all tumoral cells in both GHomas and NFPAs. In normal tissues, CXCR4 and SDF1 were expressed only in a subset of anterior pituitary cells, with a lower expression of SDF1 compared with its cognate receptor. CXCR4 and SDF1 were not confined to a specific cell population in the anterior pituitary but colocalized with discrete subpopulations of GH-, prolactin-, and adrenocorticorticotropic hormone–secreting cells. Conversely, most of the SDF1-containing cells expressed CXCR4. In six of eight pituitary adenoma primary cultures, SDF1 induced a statistically significant increase in DNA synthesis that was prevented by the treatment with the CXCR4 antagonist AMD3100 or somatostatin.Conclusions: CXCR4 and SDF1 are overexpressed in human pituitary adenomas and CXCR4 activation may contribute to pituitary cell proliferation and, possibly, to adenoma development `in humans.

Background Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. Methods We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). Results We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. Conclusions These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.

First-generation somatostatin receptor ligands (fg-SRLs), such as octreotide (OCT), represent the first-line medical therapy in acromegaly. Fg-SRLs show a preferential binding affinity for somatostatin receptor subtype-2 (SST2), while the second-generation ligand, pasireotide (PAS), has high affinity for multiple SSTs (SST5 &gt, SST2 &gt, SST3 &gt, SST1). Whether PAS acts via SST2 in somatotroph tumors, or through other SSTs (e.g., SST5), is a matter of debate. In this light, the combined treatment OCT+PAS could result in additive/synergistic effects. We evaluated the efficacy of OCT and PAS (alone and in combination) on growth hormone (GH) secretion in primary cultures from human somatotroph tumors, as well as on cell proliferation, intracellular signaling and receptor trafficking in the rat GH4C1 cell line. The results confirmed the superimposable efficacy of OCT and PAS in reducing GH secretion (primary cultures), cell proliferation, cAMP accumulation and intracellular [Ca2+] increase (GH4C1 cells), without any additive effect observed for OCT+PAS. In GH4C1 cells, co-incubation with a SST2-selective antagonist reversed the inhibitory effect of OCT and PAS on cell proliferation and cAMP accumulation, while both compounds resulted in a robust internalization of SST2 (but not SST5). In conclusion, OCT and PAS seem to act mainly through SST2 in somatotroph tumor cells in vitro, without inducing any additive/synergistic effect when tested in combination.

Mesenchymal stem cells (MSCs) can be isolated from bone marrow or other adult tissues (adipose tissue, dental pulp, amniotic fluid, and umbilical cord). In vitro, MSCs grow as adherent cells, display fibroblast‐like morphology, and self‐renew, undergoing specific mesodermal differentiation. High heterogeneity of MSCs from different origin, and differences in preparation techniques, make difficult to uniform their functional properties for therapeutic purposes. Immunomodulatory, migratory, and differentiation ability, fueled clinical MSC application in regenerative medicine, whereas beneficial effects are currently mainly ascribed to their secretome and extracellular vesicles. MSC translational potential in cancer therapy exploits putative anti‐tumor activity and inherent tropism toward tumor sites to deliver cytotoxic drugs. However, controversial results emerged evaluating either the therapeutic potential or homing efficiency of MSCs, as both antitumor and protumor effects were reported. Glioblastoma (GBM) is the most malignant brain tumor and its development and aggressive nature is sustained by cancer stem cells (CSCs) and the identification of effective therapeutic is required. MSC dualistic action, tumor‐promoting or tumor‐targeting, is dependent on secreted factors and extracellular vesicles driving a complex cross talk between MSCs and GBM CSCs. Tumor‐tropic ability of MSCs, besides providing an alternative therapeutic approach, could represent a tool to understand the biology of GBM CSCs and related paracrine mechanisms, underpinning MSC‐GBM interactions. In this review, recent findings on the complex nature of MSCs will be highlighted, focusing on their elusive impact on GBM progression and aggressiveness by direct cell‐cell interaction and via secretome, also facing the perspectives and challenges in treatment strategies., Diagrammatic representation of the interactions between mesenchymal stem cells and glioblastoma cells, involving also the other nonmalignant stromal and immune cells within the tumor microenvironment. In the figure are highlighted the critical pathways that may support or impair tumor growth via a variety of mechanisms

Glioblastoma (GBM), a high-grade glioma (WHO grade IV), is the most aggressive form of brain cancer. Available treatment options for GBM involve a combination of surgery, radiation and chemotherapy but result in a poor survival outcome. GBM is a high-vascularized tumor and antiangiogenic drugs are widely used in GBM therapy as adjuvants to control abnormal vasculature. Vasculogenic mimicry occurs in GBM as an alternative vascularization mechanism, providing a means whereby GBM can escape anti-angiogenic therapies. Here, using an in vitro tube formation assay on Matrigel&reg, we evaluated the ability of different histone deacetylase inhibitors (HDACis) to interfere with vasculogenic mimicry. We found that vorinostat (SAHA) and MC1568 inhibit tube formation by rat glioma C6 cells. Moreover, at sublethal doses for GBM cells, SAHA, trichostatin A (TSA), entinostat (MS275), and MC1568 significantly decrease tube formation by U87MG and by patient-derived human GBM cancer stem cells (CSCs). The reduced migration and invasion of HDACis-treated U87 cells, at least in part, may account for the inhibition of tube formation. In conclusion, our results indicate that HDACis are promising candidates for blocking vascular mimicry in GBM.

We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E2 (PGE2) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity.

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.

Adiponectin (Acrp30) is an adipocyte-secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co-expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87-MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches. J. Cell. Physiol. 229: 1444–1454, 2014. © 2014 Wiley Periodicals, Inc.

a b s t r a c t Cancer stem cells (CSCs) or tumor initiating cells (TICs) drive glioblastoma (GBM) development, invasive- ness and drug resistance. Distinct molecular pathways might regulate CSC biology as compared to cells in the bulk tumor mass, representing potential therapeutic targets. Chemokine CXCL12 and its receptor CXCR4 control proliferation, invasion and angiogenesis in GBM cell lines and primary cultures, but little is known about their activity in GBM CSCs. We demonstrate that CSCs, isolated from five human GBMs, express CXCR4 and release CXCL12 in vitro, although different levels of expression and secretion were observed in individual cultures, as expected for the heterogeneity of GBMs. CXCL12 treatment induced Akt-mediated significant pro-survival and self-renewal activities, while proliferation was induced at low extent. The role of CXCR4 signaling in CSC survival and self-renewal was further demonstrated using the CXCR4 antagonist AMD3100 that reduced self-renewal and survival with greater efficacy in the cultures that released higher CXCL12 amounts. The specificity of CXCL12 in sustaining CSC survival was demonstrated by the lack of AMD3100-dependent inhibition of viability in differentiated cells derived from the same GBMs. These findings, although performed on a limited number of tumor samples, suggest that the CXCL12/CXCR4 interaction mediates survival and self-renewal in GBM CSCs with high selectivity, thus emerging as a candidate system responsible for maintenance of cancer progenitors, and providing survival benefits to the tumor.

Peptide receptors involved in pathophysiological processes represent promising therapeutic targets. Neuropeptide somatostatin (SST) is produced by specialized cells in a large number of human organs and tissues. SST primarily acts as inhibitor of endocrine and exocrine secretion via the activation of five G-protein-coupled receptors, named sst1–5, while in central nervous system, SST acts as a neurotransmitter/neuromodulator, regulating locomotory and cognitive functions. Critical points of SST/SST receptor biology, such as signaling pathways of individual receptor subtypes, homo- and heterodimerization, trafficking, and cross-talk with growth factor receptors, have been extensively studied, although functions associated with several pathological conditions, including cancer, are still not completely unraveled. Importantly, SST exerts antiproliferative and antiangiogenic effects on cancer cells in vitro, and on experimental tumors in vivo. Moreover, SST agonists are clinically effective as antitumor agents for pituitary adenomas and gastro-pancreatic neuroendocrine tumors. However, SST receptors being expressed by tumor cells of various tumor histotypes, their pharmacological use is potentially extendible to other cancer types, although to date no significant results have been obtained. In this paper the most recent findings on the expression and functional roles of SST and SST receptors in tumor cells are discussed.

Chemokines control homing and trafficking of leukocytes in bone marrow and lymphoid organs. In particular, CXCL12 and its receptors CXCR4/CXCR7 control the homeostasis of multiple organs and systems. Their overexpression is linked to tumor development, both through a direct modulation of neoplastic cell proliferation, survival, and migration, and, indirectly, acting on the tumor microenvironment which sustains drug resistant tumor stem-like cells. Leukemia and lymphomas frequently display upregulation of CXCL12/CXCR4 in bone marrow that nurtures tumor cells, and confers resistance to conventional chemotherapy, increasing disease relapse. Areas covered: The authors review the molecular and cellular mechanisms by which the CXCL12/CXCR4-7 system supports leukemic bone marrow and how it contributes to leukemia development, and their potential pharmacological targeting. Besides receptor antagonists that directly inhibit leukemic cell proliferation, preclinical and clinical studies demonstrate that CXCR4 inhibition mobilizes leukemic-lymphoma cells from their niches, improving conventional chemotherapy efficacy. Clinically available and experimental pharmacological tools targeting CXCR4/CXCR7 are also described. Expert opinion: Studies have revealed the therapeutic efficacy of combining CXCR4 inhibitors and cytotoxic agents to sensitize leukemic cells, and overcome natural or acquired resistance. However, several issues are still to be unveiled (for example the role of CXCR7) to maximize therapeutic response and reduce potential toxicities.

Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

Somatostatin receptors (SSTR1–5) mediate antiproliferative effects. In C6 rat glioma cells, somatostatin is cytostatic in vitro via phosphotyrosine phosphatase-dependent inhibition of ERK1/2 activity mediated by SSTR1, -2, and -5. Here we analyzed the effects of SSTR activation on C6 glioma growth in vivo and the intracellular mechanisms involved, comparing somatostatin effects with selective agonists for SSTR1, -2, and -5 (BIM-23745, BIM-23120, BIM-23206) or receptor biselective compounds (SSTR1 and -2, BIM-23704; and SSTR2 and -5, BIM-23190). Nude mice subcutaneously xenografted with C6 cells were treated with somatostatin, SSTR agonists (50 μg, twice/day), or vehicle. Tumor growth was evaluated every 3 days for 19 days. The intracellular pathways responsible of SSTR effects in vivo were evaluated measuring Ki-67, phospho-ERK1/2, and p27kip1 expression by immunohistochemistry in sections from explanted tumors. Somatostatin and SSTR1, -2, and -5 agonists strongly inhibited in vivo C6 tumor growth, intratumoral neovessel formation, Ki-67 expression, and ERK1/2 phosphorylation and induced upregulation of p27Kip1, whereas only a modest activation of caspase-3 was observed. Somatostatin (acting on SSTR1, -2, and -5) displayed the highest efficacy; SSTR5 selective agonist showed a stronger effect than SSTR1 agonist, and SSTR2 agonist was less effective. On the other hand, SSTR1 and -2 agonists maximally reduced tumor neovascularization. The combined activation of SSTR1 and -2 showed a synergistic activity, reaching a higher efficacy than BIM-23206, whereas the simultaneous activation of SSTR2 and -5 resulted in a response resembling SSTR5 effects. Thus the simultaneous activation of different SSTRs inhibits glioma cell proliferation in vivo through both direct cytotostatic and antiangiogenic effects.

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Somatostatin inhibits cell proliferation through the activation of five receptors (SSTR1–5) expressed in normal and cancer cells. We analyzed the role of individual SSTRs in the antiproliferative activity of somatostatin in C6 rat glioma cells. Somatostatin dose-dependently inhibited C6 proliferation, an effect mimicked, with different efficacy or potency, by BIM-23745, BIM-23120, BIM-23206 (agonists for SSTR1, -2, and -5) and octreotide. The activation of SSTR3 was ineffective, although all SSTRs are functionally active, as demonstrated by the inhibition of cAMP production. All SSTRs induced cytostatic effects through the activation of the phosphotyrosine phosphatase PTPη and the inhibition of ERK1/2. For possible synergism between SSTR subtypes, we tested the effects of the combined treatment with two agonists (SSTR1+2 or SSTR2+5) or bifunctional compounds. The simultaneous activation of SSTR1 and SSTR2 slightly increased the efficacy of the individual compounds with an IC50 in between the single receptor activation. SSTR2+5 activation displayed a pattern of response superimposable to that of the SSTR5 agonist alone (low potency and higher efficacy, as compared with BIM-23120). The simultaneous activation of SSTR1, -2, and -5 resulted in a response similar to somatostatin. In conclusion, the cytostatic effects of somatostatin in C6 cells are mediated by the SSTR1, -2, and -5 through the same intracellular pathway: activation of PTPη and inhibition of ERK1/2 activity. Somatostatin is more effective than the individual agonists. The combined activation of SSTR1 and -2 shows a partial synergism as far as antiproliferative activity, whereas SSTR2 and -5 activation results in a response resembling the SSTR5 effects.

The coordinated activity of estrogens and epidermal growth factor receptor (EGFR) family agonists represents the main determinant of breast cancer cell proliferation. Stromal cell-derived factor-1 (SDF-1) enhances extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity via the transactivation of EGFR and 17beta-estradiol (E2) induces SDF-1 production to exert autocrine proliferative effects. On this basis, we evaluated whether the inhibition of the tyrosine kinase (TK) activity of EGFR may control different mitogenic stimuli in breast tumors using the EGFR-TK inhibitor gefitinib to antagonize the proliferation induced by E2 in T47D human breast cancer cells. EGF, E2, and SDF-1 induced a dose-dependent T47D cell proliferation, that being nonadditive suggested the activation of common intracellular pathways. Gefitinib treatment inhibited not only the EGF-dependent proliferation and ERK1/2 activation but also the effects of SDF-1 and E2, suggesting that these activities were mediated by EGFR transactivation. Indeed, both SDF-1 and E2 caused EGFR tyrosine phosphorylation. The molecular link between E2 and SDF-1 proliferative effects was identified because 1,1'-(1,4-phenylenebis(methylene))-bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist, inhibited SDF-1- and E2-dependent proliferation and EGFR and ERK1/2 phosphorylation. EGFR transactivation was dependent on c-Src activation. E2 treatment caused a powerful SDF-1 release from T47D cells. Finally, in SKBR3, E2-resistant cells, EGFR was constitutively activated, and AMD3100 reduced EGFR phosphorylation and cell proliferation, whereas HER2-neu was transactivated by SDF-1 in SKBR3 but not in T47D cells. In conclusion, we show that activation of CXCR4 transduces proliferative signals from the E2 receptor to EGFR, whose inhibition is able to revert breast cancer cell proliferation induced by multiple receptor activation.

Chemokines participate in cellular processes associated with tumor proliferation, migration, and angiogenesis. We previously demonstrated that stromal cell–derived factor 1 (SDF1) exerts a mitogenic activity in glioblastomas through the activation of its receptor CXCR4. Here we studied the expression of this chemokine in human meningiomas and its possible role in cell proliferation. Reverse transcriptase–PCR analysis for CXCR4 and SDF1 was performed on 55 human meningiomas (47 WHO grade I, 5 WHO II, and 3 WHO III). Immunolabeling for CXCR4 and SDF1 was performed on paraffin-embedded sections of these tumors. [3H]Thymidine uptake and Western blot analyses were performed on primary meningeal cell cultures of tumors to evaluate the proliferative activity of human SDF1α (hSDF1α) in vitro and the involvement of extracellular signal–regulated kinase 1/2 (ERK1/2) activation in this process. CXCR4 mRNA was expressed by 78% of the tumor specimens and SDF1 mRNA by 53%. CXCR4 and SDF1 were often detected in the same tumor tissues and colocalized with epithelial membrane antigen immunostaining. In 9 of 12 primary cultures from meningiomas, hSDF1α induced significant cell proliferation that was strongly reduced by the mitogen-activated protein kinase kinase inhibitor PD98059, involving ERK1/2 activation in the proliferative signal of hSDF1α. In fact, CXCR4 stimulation led to ERK1/2 phosphorylation/activation. In addition, the hSDF1α-induced cell proliferation was significantly correlated with the MIB1 staining index in the corresponding surgical specimen. In conclusion, we found that human meningiomas express CXCR4 and SDF1 and that hSDF1α induces proliferation in primary meningioma cell cultures through the activation of ERK1/2.

// Alessandro Corsaro 1 , Adriana Bajetto 1 , Stefano Thellung 1, 2 , Giulia Begani 1 , Valentina Villa 1 , Mario Nizzari 1 , Alessandra Pattarozzi 1 , Agnese Solari 1 , Monica Gatti 1 , Aldo Pagano 3, 4 , Roberto Wurth 1 , Antonio Daga 4 , Federica Barbieri 1, 2 , Tullio Florio 1, 2 1 Sezione di Farmacologia, Dipartimento di Medicina Interna, University of Genova, Genova, Italy 2 Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genova, Italy 3 Department of Experimental Medicine, University of Genova, Genova, Italy 4 IRCCS AOU San Martino - IST, Genova, Italy Correspondence to: Tullio Florio, email: tullio.florio@unige.it Keywords: prion protein, glioblastoma, cancer stem cells, in vivo tumorigenicity, GFAP Received: December 30, 2015 Accepted: May 04, 2016 Published: May 24, 2016 ABSTRACT Prion protein (PrP C ) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrP C is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrP C regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrP C in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrP C expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrP C expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrP C down-regulated cells. Moreover, PrP C down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways ( i.e. increased GFAP expression). Our results suggest that PrP C controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Drug repositioning is gaining increasing attention in drug discovery because it represents a smart way to exploit new molecular targets of a known drug or target promiscuity among diverse diseases, for medical uses different from the one originally considered. In this review, we focus on known non-oncological drugs with new therapeutic applications in oncology, explaining the rationale behind this approach and providing practical evidence. Moving from incompleteness of the knowledge of drug-target interactions, particularly for older molecules, we highlight opportunities for repurposing compounds as cancer therapeutics, underling the biologically and clinically relevant affinities for new targets. Ideal candidates for repositioning can contribute to the therapeutically unmet need for more-efficient anticancer agents, including drugs that selectively target cancer stem cells.

Chemokines are a family of proteins that have pleiotropic biological effects. They are well known to regulate the recruitment and trafficking of leukocytes to sites of inflammation. Chemokines are grouped into four classes based on the positions of key cysteine residues: C, CC, CXC, and CX3C. Stromal cell-derived factor-1 (SDF-1), the ligand of the CXCR4 receptor, is a CXC chemokine and is a highly conserved gene. Ovarian cancer typically disseminates widely in the abdomen, a characteristic that limits curative therapy. The mechanisms that promote ovarian cancer proliferation are incompletely understood. We studied a human ovarian adenocarcinoma cell line (OC 314) and investigated the role of CXCR4 activation by SDF-1 in human ovarian cancer. We demonstrate that CXCR4 and SDF-1 mRNA are expressed in OC 314. We show that SDF-1alpha induces proliferation in ovarian cancer cells in a dose-dependent manner. Moreover, we demonstrate that the SDF-1-dependent proliferation correlates to the phosphorylation and activation of extracellular signal-regulated kinases (ERK)1/2, which in turn are correlated to epidermal growth factor (EGF) receptor transactivation. In fact, AG1478, a specific inhibitor of the EGF receptor kinase, blocked both SDF-1alpha-dependent proliferation and ERK1/2 activation.

Pyrrolidinedithiocarbamate (PDTC) is a compound displaying antioxidant, pro-oxidant and metal chelator properties in different cell types. It has been described that PDTC may exert either anti-apoptotic or apoptotic activity. Moreover it is known that this agent regulates the activity of redox-sensitive transcription factors, such as AP-1 and NF-kappaB. Using cerebellar granule cells (CGCs), a well-described model of neuronal primary cultures, we investigated the effects of different concentrations of this compound on cell viability and the intracellular mechanisms involved. PDTC used at concentrations, as low as 1 microM, exerts cytotoxic effects on CGC through the activation of the apoptotic machinery with a maximal efficacy for concentration of 10 microM. The PDTC-dependent apoptosis is correlated to a biphasic and long-lasting increase of AP-1 binding to the DNA, apparently without affecting the NF-kappaB whose activity was reduced only at much higher concentrations (100 microM). PDTC treatment enhanced ERK phosphorylation (maximal effect 1h) and p38 phosphorylation (maximal effect 7h) that was accompanied by an increase of both mRNA and protein of c-Jun. In conclusion the results presented show that PDTC exerts apoptotic effects on CGC, that are correlated to the activation of stress-pathways, involving mainly AP-1 and MAPKs.

Chemokines represent key factors in the outburst of the immune response, by activating and directing the leukocyte traffic, both in lymphopoiesis and in immune surveillance. Neurobiologists took little interest in chemokines for many years, until their link to acquired immune deficiency syndrome-associated dementia became established, and thus their importance in this field has been neglected. Nevertheless, the body of data on their expression and role in the CNS has grown in the past few years, along with a new vision of brain as an immunologically competent and active organ. A large number of chemokines and chemokine receptors are expressed in neurons, astrocytes, microglia and oligodendrocytes, either constitutively or induced by inflammatory mediators. They are involved in many neuropathological processes in which an inflammatory state persists, as well as in brain tumor progression and metastasis. Moreover, there is evidence for a crucial role of CNS chemokines under physiological conditions, similar to well known functions in the immune system, such as proliferation and developmental patterning, but also peculiar to the CNS, such as regulation of neural transmission, plasticity and survival.

Chemokines are crucial autocrine and paracrine players in tumor development. In particular, CXCL12, through its receptors CXCR4 and CXCR7, affects tumor progression by controlling cancer cell survival, proliferation and migration, and, indirectly, via angiogenesis or recruiting immune cells.Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults and despite current multimodal therapies it remains almost incurable. The aggressive and recurrent phenotype of GBM is ascribed to high growth rate, invasiveness to normal brain, marked angiogenesis, ability to escape the immune system and resistance to standard of care therapies. Tumor molecular and cellular heterogeneity severely hinders GBM therapeutic improvement. In particular, a subpopulation of chemo- and radio-therapy resistant tumorigenic cancer stem–like cells (CSCs) is believed to be the main responsible for tumor cell dissemination to the brain.GBM cells display heterogeneous expression levels of CXCR4 and CXCR7 that are overexpressed in CSCs, representing a molecular correlate for the invasive potential of GBM. The microenvironment contribution in GBM development is increasingly emphasized. An interplay exists between CSCs, differentiated GBM cells, and the microenvironment, mainly through secreted chemokines (e.g. CXCL12) causing recruitment of fibroblasts, endothelial, mesenchymal and inflammatory cells to the tumor, via specific receptors such as CXCR4.This review covers recent developments on the role of CXCL12/CXCR4-CXCR7 networks in GBM progression and the potential translational impact of their targeting. The biological and molecular understanding of the heterogeneous GBM cell behavior, phenotype and signaling is still limited. Progress in the identification of chemokine-dependent mechanisms that affect GBM cell survival, trafficking and chemo-attractive functions, opens new perspectives for development of more specific therapeutic approaches that include chemokine-based drugs.

Stromal cell-derived factor-1 (SDF-1), the ligand of the CXCR4 receptor, is a chemokine involved in chemotaxis and brain development that also acts as co-receptor for HIV-1 infection. We previously demonstrated that CXCR4 and SDF-1alpha are expressed in cultured type-I cortical rat astrocytes, cortical neurones and cerebellar granule cells. Here, we investigated the possible functions of CXCR4 expressed in rat type-I cortical astrocytes and demonstrated that SDF-1alpha stimulated the proliferation of these cells in vitro. The proliferative activity induced by SDF-1alpha in astrocytes was reduced by PD98059, indicating the involvement of extracellular signal-regulated kinases (ERK1/2) in the astrocyte proliferation induced by CXCR4 stimulation. This observation was further confirmed showing that SDF-1alpha treatment selectively activated ERK1/2, but not p38 or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Moreover, both astrocyte proliferation and ERK1/2 phosphorylation, induced by SDF-1alpha, were inhibited by pertussis toxin (PTX) and wortmannin treatment indicating the involvement of a PTX sensitive G-protein and of phosphatidyl inositol-3 kinase in the signalling of SDF-1alpha. In addition, Pyk2 activation represent an upstream components for the CXCR4 signalling to ERK1/2 in astrocytes. To our knowledge, this is the first report demonstrating a proliferative effect for SDF-1alpha in primary cultures of rat type-I astrocytes, and showing that the activation of ERK1/2 is responsible for this effect. These data suggest that CXCR4/SDF-1 should play an important role in physiological and pathological glial proliferation, such as brain development, reactive gliosis and brain tumour formation.

Chemokines are a family of proteins associated with the trafficking of leukocytes in physiological immune surveillance and inflammatory cell recruitment in host defence. They are classified into four classes based on the positions of key cystiene residues: C, CC, CXC, and CX3C. Chemokines act through both specific and shared receptors that all belong to the superfamily of G-protein-coupled receptors. Besides their well-established role in the immune system, several recent reports have demonstrated that these proteins also play a role in the central nervous system (CNS). In the CNS, chemokines are constitutively expressed by microglial cells, astrocytes, and neurons, and their expression can be increased after induction with inflammatory mediators. Constitutive expression of chemokines and chemokine receptors has been observed in both developing and adult brains, and the role played by these proteins in the normal brain is the object of intense study by many research groups. Chemokines are involved in brain development and in the maintenance of normal brain homeostasis; these proteins play a role in the migration, differentiation, and proliferation of glial and neuronal cells. The chemokine stromal cell-derived factor 1 and its receptor, CXCR4, are essential for life during development, and this ligand-receptor pair has been shown to have a fundamental role in neuron migration during cerebellar formation. Chemokine and chemokine receptor expression can be increased by inflammatory mediators, and this has in turn been associated with several acute and chronic inflammatory conditions. In the CNS, chemokines play an essential role in neuroinflammation as mediators of leukocyte infiltration. Their overexpression has been implicated in different neurological disorders, such as multiple sclerosis, trauma, stroke, Alzheimer's disease, tumor progression, and acquired immunodeficiency syndrome-associated dementia. An emerging area of interest for chemokine action is represented by the communication between the neuroendocrine and the immune system. Chemokines have hormone-like actions, specifically regulating the key host physiopathological responses of fever and appetite. It is now evident that chemokines and their receptors represent a plurifunctional family of proteins whose actions on the CNS are not restricted to neuroinflammation. These molecules constitute crucial regulators of cellular communication in physiological and developmental processes.

Human immunodeficiency virus (HIV-1) infects the brain and causes a progressive encephalopathy in 20 to 30% of infected children and adults called AIDS dementia complex. Evidence from in vitro and in vivo studies suggests a role for the viral envelope glycoprotein gp120, as a mediator of neurotoxicity. However, the site of interaction of gp120 with neurons and astrocytes to mediate neuronal death is still unknown. Recently the chemokine receptors, CCR5 and CXCR4, have been identified as co-receptors together with CD4 for HIV-1 entry into the target cells, suggesting a possible role for these receptors in the pathogenesis of the HIV-1 infection in the brain. Here we report the expression of CCR5 and CXCR4 in many different rat brain areas. We also found both receptors in cultured type I astrocytes demonstrating that glial cells may represent an important target for chemokines in vivo. Indeed, the functional capacity of CXCR4 receptor in astrocytes was demonstrated showing that SDF 1 alpha induced an increase of intracellular calcium concentration.

In this work we studied the subcellular localization of ciliary neurotrophic factor (CNTF) in primary culture of rat cortical type I astrocytes and rat glioma C6 cells, transfected COS-7 cells and in the Xenopus oocytes. In all these models, morphological and biochemical evidence are provided for the nuclear localization of CNTF. In addition the nuclear translocation of CNTF is temperature-sensitive and thus strongly suggestive of a mechanism of facilitated transport.

HIV-1 infection frequently induces neuronal death responsible for the development of neurological deficits associated with AIDS. Several reports suggest that gp120, the HIV-1 envelope glycoprotein, is the main candidate as mediator of the HIV-1-dependent neurotoxicity. Here we report the effect of gp120 on the survival of cortical neurons in vitro and the possible mechanisms whereby it occurs. Mature cortical neurons, cultured on a feeder layer of astrocytes, were treated with gp120 in a defined culture medium in absence of serum. The treatment with gp120 induced time-dependent neuronal damage displaying apoptotic features, as revealed by in situ labelling of DNA fragmentation. TGF-β1, a cytokine that has been previously shown to exert neuroprotective effects, prevented the cell death induced by exposure of cortical neurons to gp120. The prolonged treatment with gp120 also increased neuronal [Ca2+]i, while the coincubation with TGF-β1 completely prevented the impairment of neuronal Ca2+ homeostasis. These data, taken together, demonstrate that gp120 induces apoptosis in cortical neurons, an effect that can be related to the impairment of Ca2+ homeostasis, and that TGF-β1 pretreatment reverts both the neuronal death and the alterations in neuronal [Ca2+]i. J. Neurosci. Res. 49:600–607, 1997. © 1997 Wiley-Liss, Inc.

IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family, isolated from supernatants of Ig stimulated monocytes, that binds competitively to IL-1 receptors without stimulating target cells (1-3). Also epithelial cells produce IL-1ra in a form which lacks a secretory signal sequence (4).Here we have compared the biosynthesis and secretion of IL-1ra in monocytes and keratinocytes. Our data show that monocytes produce two molecular forms of IL-1ra, of 18 Kd and 23 Kd respectively, which differ in the degree of glycosylation. Both forms are secreted via the "classical" endoplasmic reticulum (ER)-Golgi secretory pathway. By contrast keratinocytes produce IL-1ra in a molecular form of 20 Kd, which is not N-glycosylated: 20 Kd IL-1ra is detectable in supernatants of keratinocytes, although in small amounts. The presence of IL-1ra in keratinocytes cultures fluids is not inhibited by Brefeldin A (BFA), suggesting a possible secretion through the leaderless secretory pathway.

Malignant pleural mesothelioma (MPM) is an aggressive chemotherapy-resistant cancer. Up-regulation of epidermal growth factor receptor (EGFR) plays an important role in MPM development and EGFR-tyrosine kinase inhibitors (TKIs) may represent novel therapeutic options. We tested the effects of the EGFR TKIs gefitinib and erlotinib and TKIs targeted to other growth factors (VEGFR and PDGFR), in comparison to standard antineoplastic agents, in two human MPM cell lines, IST-Mes2 and ZL55. All drugs showed IC(50) values in the micromolar range: TKIs induced cytostatic effects at concentrations up to the IC(50,) while conventional drug growth-inhibitory activity was mainly cytotoxic. Moreover, the treatment of IST-Mes2 with TKIs (gefitinib and imatinib mesylate) in combination with cisplatin and gemcitabine did not show additivity. Focusing on the molecular mechanisms underlying the antiproliferative and pro-apoptotic effects of EGFR-TKIs, we observed that gefitinib induced the formation and stabilization of inactive EGFR homodimers, even in absence of EGF, as demonstrated by EGFR B(max) and number of sites/cell. The analysis of downstream effectors of EGFR signaling demonstrated that EGF-induced proliferation, reverted by gefitinib, involved ERK1/2 activation, independently from Akt pathway. Gefitinib inhibits MPM cell growth and survival, preventing EGF-dependent activation of ERK1/2 pathway by blocking EGFR-TK phosphorylation and stabilizing inactive EGFR dimers. Along with the molecular definition of TKIs pharmacological efficacy in vitro, these results may contribute to delve deep into the promising but still controversial role for targeted and conventional drugs in the therapy of MPM.

Glioblastoma (GBM) is the most common and aggressive neoplasia of the central nervous system in adults. Despite continued improvements in surgery, chemotherapy, and radiotherapy, clinical outcome is still dismal and better understanding of GBM biology is needed to develop novel therapies. Recent studies have demonstrated the existence of a small subpopulation of cells with stem-like features termed cancer stem cells (CSCs) in several human cancers, including GBM. CSCs are slow growing, self-renewable and highly tumorigenic, give rise to progeny of multiple lineages, and are chemo-radio-resistant, often expressing high levels of multidrug resistance and drug efflux genes. According to CSC hypothesis, current therapies are cytotoxic to the bulk of highly proliferative tumor cells but fail to kill the relatively quiescent and resistant CSC subpopulation, thus allowing these cells to survive and induce tumor recurrence. These characteristics allow GBM CSCs to survive cytotoxic therapies and drive tumor recurrence. The epidermal growth factor receptor (EGFR/HER1) belongs to the receptor tyrosine kinase (TK) family that regulates cell proliferation, survival, differentiation and motility. Overexpression of EGFR occurs in several tumors, including GBM, correlates with increased cell proliferation, decreased apoptosis, and a poorer prognosis, sustaining cancer development and progression. Small-molecules targeting EGFR-TK (TK inhibitors, TKIs) are the most clinically developed EGFR targeted-therapies for the treatment of GBM. We reported that cultures enriched in CSC isolated from human GBMs undergo growth arrest and cell death in the presence of EGFR-TKIs. The high incidence of EGFR overexpression, amplification or co-expression of the mutated, constitutively active EGFRvIII in GBMs raised expectations that treatment with EGFR TKIs, such as gefitinib or erlotinib, would have significant positive effects. This review summarizes current knowledge regarding EGFR molecular abnormalities and dysregulation in high-grade gliomas, the role of CSCs in GBM, and discusses the implications of the CSC hypothesis for the development of future EGFR-targeted therapies for brain tumors.

Chemokines are small secreted proteins that chemoattract and activate immune and non-immune cells. Their role in the immune system is well-known, and it has recently been suggested that they may also play a role in the central nervous system (CNS). Indeed, they do not only act as immunoinflammatory mediators in the brain but they also act as potential modulators in neurotransmission. Although we are only beginning to be aware of the implication of chemokines in neuroendocrine functions, this review aims at summarizing what is known in that booming field of research. First we describe the expression of chemokines and their receptors in the CNS with a focus on the hypothalamo-pituitary system. Secondly, we present what is known on some chemokines in the regulation of neuroendocrine functions such as cell migration, stress, thermoregulation, drinking and feeding as well as anterior pituitary functions. We suggest that chemokines provide a fine modulatory tuning system of neuroendocrine regulations.

Ovarian cancer is the most common type of gynecologic malignancy. Despite advances in surgery and chemotherapy, the survival rate is still low since most ovarian cancers relapse and become drug-resistant. Chemokines are small chemoattractant peptides mainly involved in the immune responses. More recently, chemokines were also demonstrated to regulate extra-immunological functions. It was shown that the chemokine network plays crucial functions in the tumorigenesis in several tissues. In particular the imbalanced or aberrant expression of CXCL12 and its receptor CXCR4 strongly affects cancer cell proliferation, recruitment of immunosuppressive cells, neovascularization, and metastasization. In the last years, several molecules able to target CXCR4 or CXCL12 have been developed to interfere with tumor growth, including pharmacological inhibitors, antagonists, and specific antibodies. This chemokine ligand/receptor pair was also proposed to represent an innovative therapeutic target for the treatment of ovarian cancer. Thus, a thorough understanding of ovarian cancer biology, and how chemokines may control these different biological activities might lead to the development of more effective therapies. This paper will focus on the current biology of CXCL12/CXCR4 axis in the context of understanding their potential role in ovarian cancer development.

Purpose: Hypothalamic or locally produced growth factors and cytokines control pituitary development, functioning, and cell division. We evaluated the expression of the chemokine stromal cell–derived factor 1 (SDF1) and its receptor CXCR4 in human pituitary adenomas and normal pituitary tissues and their role in cell proliferation. Experimental Design: The expression of SDF1 and CXCR4 in 65 human pituitary adenomas and 4 human normal pituitaries was determined by reverse transcription-PCR, immunohistochemistry, and confocal immunofluorescence. The proliferative effect of SDF1 was evaluated in eight fibroblast-free human pituitary adenoma cell cultures. Results: CXCR4 mRNA was expressed in 92% of growth hormone (GH)-secreting pituitary adenomas (GHoma) and 81% of nonfunctioning pituitary adenomas (NFPA), whereas SDF1 was identified in 63% and 78% of GHomas and NFPAs, respectively. Immunostaining for CXCR4 and SDF1 showed a strong homogenous labeling in all tumoral cells in both GHomas and NFPAs. In normal tissues, CXCR4 and SDF1 were expressed only in a subset of anterior pituitary cells, with a lower expression of SDF1 compared with its cognate receptor. CXCR4 and SDF1 were not confined to a specific cell population in the anterior pituitary but colocalized with discrete subpopulations of GH-, prolactin-, and adrenocorticorticotropic hormone–secreting cells. Conversely, most of the SDF1-containing cells expressed CXCR4. In six of eight pituitary adenoma primary cultures, SDF1 induced a statistically significant increase in DNA synthesis that was prevented by the treatment with the CXCR4 antagonist AMD3100 or somatostatin. Conclusions: CXCR4 and SDF1 are overexpressed in human pituitary adenomas and CXCR4 activation may contribute to pituitary cell proliferation and, possibly, to adenoma development `in humans.

Stromal cell-derived factor-1 (SDF-1) is a chemokine of the CXC subfamily that exerts its effects via CXCR4, a G-protein-coupled receptor. CXCR4 is often expressed by tumor cells, and its activation causes tumor cell proliferation. Using GH4C1 cells, here we show that SDF-1 induced cell proliferation in a dose-dependent manner. Thus, we evaluated the intracellular signaling involved in this effect. SDF-1 increased cytosolic [Ca2+] and activated Pyk2, ERK1/2, and BKCa channels. To correlate these intracellular effectors with the proliferative activity of SDF-1, we inhibited their activity using BAPTA-AM (Ca2+ chelator), PD98059 (MEK inhibitor), salicylate (Pyk2 inhibitor), and TEA (K+ channel blocker). All these compounds reverted SDF-1-induced proliferation, suggesting the involvement of multiple intracellular pathways. To identify a possible crosstalk and a molecular ordering among these pathways, we tested these antagonists on SDF-1-dependent activation of ERK1/2, Pyk2, and BKCa channels. We report that the inhibition of [Ca2+]i increase or the blockade of BKCa channel activity did not affect ERK1/2 activation by SDF-1; Pyk2 activation was purely Ca2+-dependent, not involving ERK1/2 or BKCa channels; and BKCa channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that SDF-1-dependent increase of [Ca2+]i activates Pyk2, which, in turn, regulates BKCa channel activity. Conversely, ERK1/2 activation is an independent phenomenon. In conclusion, we demonstrate that SDF-1 induces proliferation of GH4C1 cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for pituitary cell proliferation which may contribute to pituitary adenoma development.

Chemokines are key factors involved in the regulation of immune response, through the activation and control of leukocyte traffic, lymphopoiesis and immune surveillance. However, a large number of chemokines and their receptors are expressed in central nervous system (CNS) cells, either constitutively or induced by inflammatory stimuli, playing a role in many neuropathological processes. Stromal cell-derived factor 1 (SDF1) is a chemokine whose extra-immunological localization and functions have been extensively studied. SDF1 and its receptor CXCR4 were identified in both neurons and glia of many brain areas, including the hypothalamus, as well as at the pituitary level. Importantly, SDF1 and CXCR4 expression is increased in brain tumors in which their activity induced tumor cell proliferation and brain parenchyma invasion. Despite their localization, to date very few reports addressed the role of CXCR4 and SDF1 in the modulation of the hypothalamus/pituitary axis and their possible involvement in the development of pituitary adenomas. In this review, we discuss previous literature data on the role of chemokines in normal and adenomatous pituitary cells, focusing on recent data from our group showing that CXCR4 activation controls proliferation and both prolactin and GH release in the pituitary adenoma cell line GH4C1 through a complex network of intracellular signals. Thus, the SDF1/CXCR4 system together with other chemokinergic ligand–receptor pairs, may represent a novel regulatory pathway for pituitary function and, possibly, be involved in pituitary adenoma development. These lines of evidence suggest that the inhibition of chemokine receptors may represent a novel pharmacological target for the treatment of pituitary adenomas.

Chemokines participate in cellular processes associated with tumor proliferation, migration, and angiogenesis. We previously demonstrated that stromal cell-derived factor 1 (SDF1) exerts a mitogenic activity in glioblastomas through the activation of its receptor CXCR4. Here we studied the expression of this chemokine in human meningiomas and its possible role in cell proliferation. Reverse transcriptase-PCR analysis for CXCR4 and SDF1 was performed on 55 human meningiomas (47 WHO grade I, 5 WHO II, and 3 WHO III). Immunolabeling for CXCR4 and SDF1 was performed on paraffin-embedded sections of these tumors. [(3)H]Thymidine uptake and Western blot analyses were performed on primary meningeal cell cultures of tumors to evaluate the proliferative activity of human SDF1alpha (hSDF1alpha) in vitro and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) activation in this process. CXCR4 mRNA was expressed by 78% of the tumor specimens and SDF1 mRNA by 53%. CXCR4 and SDF1 were often detected in the same tumor tissues and colocalized with epithelial membrane antigen immunostaining. In 9 of 12 primary cultures from meningiomas, hSDF1alpha induced significant cell proliferation that was strongly reduced by the mitogen-activated protein kinase kinase inhibitor PD98059, involving ERK1/2 activation in the proliferative signal of hSDF1alpha. In fact, CXCR4 stimulation led to ERK1/2 phosphorylation/activation. In addition, the hSDF1alpha-induced cell proliferation was significantly correlated with the MIB1 staining index in the corresponding surgical specimen. In conclusion, we found that human meningiomas express CXCR4 and SDF1 and that hSDF1alpha induces proliferation in primary meningioma cell cultures through the activation of ERK1/2.

Recent evidence indicates that cancer cells express chemokine (CK) receptors and that their signaling is crucial for tumor proliferation, migration, and angiogenesis. The profiles of expression of CXC CK receptors (CXCR1-5) and their main ligands (growth-related oncogene, GRO1-2-3/CXCL1-2-3; interleukin 8, IL-8/CXCL8; monokine-induced gamma-interferon MIG/CXCL9; gamma-interferon-inducible-protein-10, IP-10/CXCL10; stromal cell-derived factor-1, SDF1/CXCL12; B-cell activating CK-1, BCA-1/CXCL13) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) in surgical samples of human meningiomas. All the five receptors displayed high percentages of positive cases: 92% CXCR1, 89% CXCR2, 83% CXCR3, 78% CXCR4, and 94% CXCR5. Conversely, their ligands showed a lower pattern of expression: 40% IL-8, 42% GRO1-3, 42% IP-10, 28% MIG, 53% SDF1, and 3% BCA-1. SDF1/CXCR4 interaction plays a pivotal role in cancer proliferation. Thus, the signaling mechanisms activated by the exclusive binding between SDF1 and CXCR4 was investigated in 12 primary cultures from meningioma tissues. CXCR4 was functionally coupled as demonstrated by the significant increase of DNA synthesis in meningioma cells in response to SDF1, measured by [3H]-thymidine uptake. In three primary cultures, the SDF1-dependent mitogenic activity was associated with a marked phosphorylation of extracellular signal-regulated kinase (ERK1/2) as evaluated by Western blots. PD98059 (a MEK inhibitor) significantly reduced ERK1/2 activation, thus linking the SDF1/CXCR4 pathway to meningioma cell proliferation via ERK1/2 signal transduction. We demonstrate, for the first time in human meningiomas, the simultaneous expression of CXCR1-5 and their CKs and the mitogenic activity of SDF1/CXCR4, suggesting a pivotal role of these receptor-ligand pairs in meningeal tumors.

Chemokines have been involved in cellular processes associated to malignant transformation such as proliferation, migration and angiogenesis. The expression of five CXC chemokine receptors and their main ligands was analysed by RT-PCR in 31 human astrocytic neoplasms. The mRNAs for all the receptors analysed were identified in a high percentage of tumours, while their ligands showed lower expression. CXCR4 and SDF1 were the most frequently mRNA identified (29/31 and 13/31 of the gliomas studied, respectively). Thus, we further analysed the cell localization of CXCR4 and SDF1 in immunohistochemistry experiments. We show a marked co-localization of CXCR4 and SDF1 in tumour cells, mainly evident in psudolpalisade and microcystic degeneration areas and in the vascular endothelium. In addition, hSDF1alpha induced a significant increase of DNA synthesis in primary human glioblastoma cell cultures and chemotaxis in a glioblastoma cell line. These results provide evidence of the expression of multiple CXC chemokines and their receptors in brain tumours and that in particular CXCR4 and SDF1 sustain proliferation and migration of glioma cells to promote malignant progression.

We used GH4C1 cells as a model to study the effects of the chemokine stromal cell-derived factor 1 (SDF1) in pituitary functions. In these cells, SDF1alpha induced proliferation and growth hormone secretion, suggesting a possible regulatory role for this chemokine at pituitary level. We evaluated the intracellular signaling involved in these effects: SDF1alpha increased cytosolic [Ca(2+)] and activated Pyk2, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. To correlate these intracellular effectors with the proliferative and secretory effects, we inhibited their activity using BAPTA-AM (Ca(2+) chelator), 2'-amino-3'-methoxyflavone (PD98059; a mitogen-activated protein kinase kinase inhibitor), salicylate (Pyk2 inhibitor), and tetraethyl ammonium (K(+) channel blocker). All of these compounds reverted SDF1alpha-induced proliferation, suggesting the involvement of multiple intracellular pathways. Conversely, only BAPTA-AM reverted growth hormone secretion. To identify a possible cross-talk and a molecular ordering among these pathways, we tested these antagonists on SDF1alpha-dependent activation of ERK1/2, Pyk2, and BK(Ca) channels. From these experiments, we observed that the inhibition of [Ca(2+)](i) increase or BK(Ca) channel activity did not affect ERK1/2 activation by SDF1alpha; Pyk2 activation was purely Ca(2+)-dependent, not involving ERK1/2 or BK(Ca) channels; and BK(Ca) channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that an SDF1alpha-dependent increase of [Ca(2+)](i) activates Pyk2, which in turn regulates BK(Ca) channel activity. Conversely, ERK1/2 activation is an independent phenomenon. In conclusion, we demonstrate that SDF1alpha causes both proliferation and growth hormone release from pituitary adenoma cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for growth hormone secretion and pituitary cell proliferation, which may contribute to pituitary adenoma development.

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1alpha treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1alpha induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important "cross-talk" between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.

The study of chemokine role in the CNS indubitably represents an important step to understanding many aspects of brain pathology, physiology and development. Here we discuss our recent research on the expression of chemokines and chemokine receptors in brain tissues and in cultured CNS cells, with particular regard to the CXCL12/SDF-1-CXCR4 system. We showed their expression in both glial and neuronal cells in basal conditions and their modulation upon stimulation. We demonstrated that CXCL12/SDF-1 in vitro act as a growth factor for astrocytes by stimulating their proliferation, a phenomenon that could represent the basis of pathological conditions such as gliosis and malignant transformation. We investigated the signal transduction pathways, identifying in the sequential activation of G-protein-PI-3Kinase-ERK1/2 the main signaling cascade linked to the CXCL12/SDF-1-induced proliferation in astrocytes.